The Lipophilic hormones such as steroids, retinoids, and thyroid hormones can permeate a target cell and through their interaction with nuclear receptors, modify gene expression. Cloning and characterization of such receptors has shown that the binding of hormone to its receptor triggers an allosteric change thereof which in turns enables the hormone-receptor complex to specifically interact with a DNA target and modulate transcription. It is now widely recognized that these receptors are actually part of a superfamily of structurally related nuclear receptors which interact with chemically distinct ligands to directly affect gene expression. The importance of the nuclear receptor superfamily in maintenance of homeostasis and physiology of cells and organisms is demonstrated by the high level of conservation throughout evolution of the more than 150 members already characterized.
The nuclear receptors are characterized by (1) a DNA binding domain (DBD), responsible for the targeting of receptors to their specific response elements (RE); and (2) a ligand-binding domain (LBD), which ensures the specificity, selectivity and affinity of the binding of the ligand to its receptor (For reviews see, Mangelsdorf et al., 1995, Cell 83: 835-839; and Ibid 841-850). Characterization of the RE has shown that the RE consists of a core half-site defined by a degenerate Xn-AGGTCA which can be configured as direct repeats (DR), inverted repeats (IR), everted repeats (ER) or nonrepeats (NR) [PCT publication number WO96/21457 published Jul. 18, 1996]. Since the nuclear receptor recognize REs which are unique, it follows that subtle differences in the sequence of the RE or their configuration have significant effects on DNA binding of the receptor (Mangelsdorf et al., 1995, supra; and Ibid 841-850). Once bound to a RE, each receptor responds to its signal through the C-terminal ligand binding domain (LBD). The LBD contains several embedded subdomains which may include a C-terminal transactivation function, a series of heptad repeats which may serve as a dimerization interface and a poorly-delineated transcriptional suppression domain. In its natural context of the LBD, transcriptional activity through the transactivation domain, requires the addition of ligand (WO 96/21457).
A significant number of nuclear receptors are termed orphan receptors (no ligand which binds thereto has been identified). Such orphans have been identified by homology to the initial members of the superfamily in every metazoan species. It remains a significant challenge to identify a function for these orphan receptors, as well as to identify ligands and/or hormones that affect the activity thereof (Mangelsdorf et al., 1995, supra; and Ibid 841-850).
Nur77 (also known as NGFI-B, N10, NAK1, and TR3), was the first member of the Nur family of the orphan receptor subfamily of nuclear receptors to be identified. Other members of the Nur family include Nurr1 (for Nur-related member number one; also known as RNR-1, NOT and TINUR) and NOR-1 (also known as MINOR). Nur77 distinguishes itself by its ability to bind DNA as a monomer (Fahrner et al., 1991, Science 252:1296-1300; wilson et al., 1992, Science 256:107-110) and by its role in TCR-induced apoptosis in T cell (Liu et al., 1994, Nature 367:281-284; Woronicz et al., 1994, Nature 367:277-281; Calnan et al., 1995, Immunity 3:273-282). Of note, Nurr-1, the β isoform of Nur77 is described as a constitutively active orphan receptor that binds as a high-affinity monomer to an AA-AGGTCA core site and thus to the synthetic NBRE sequence (WO 96/21457). Indeed, WO 96/21457 teaches that Nurr-1 provides a well characterized example of the paradigms of binding of nuclear receptor as a monomer to a single core site.
Nur77 has been cloned repeatedly by numerous investigators either as a mitogen-inducible gene or as an immediate early gene (Hazel et al., 1988, Proc. Natl. Acad. Sci. USA 85:8444-8448; Milbrandt 1988, J. Neuron 1:183-188; Ryseck et al., 1989, EMBO J. 8:3327-3335; Nakai et al., 1990, J. Mol. Endocrinol. 4:1438-1443). Recent work has indicated that it is widely expressed, in particular throughout the brain.
Nur77 was shown to heterodimerize with RXR to confer 9-cis retinoic acid-dependent transcription (Perlmann et al., 1995, Genes Dev. 9:769-782; Forman et al., 1995, Cell 81:541-550) (WO 96/21457). Two common features of nonstemid receptors that have known ligands have been identified: the ligands are small lipophilic compounds and RXR Is part of the receptor complexes. Thus, orphan receptors such as Nur77 are likely candidates for ligand-dependent activation (Mangelsdorf et al., 1995, Cell 83: 841-850).
The experiments showing heterodimerization with RXR were carried out with two synthetic DNA elements: NBRE (Honkaniemi et al., 1994, Brain Res. 25:234-241)_(WO 96/21457) and DR-5 (Forman et al., 1995, supra). Synthetic NBRE was initially identified as a putative target for Nur77 by genetic selection in yeast (Fahrner et al., 1991, supra). Importantly in these experiments, Nur77 was shown to activate transcription as a monomer (Fahrner et al., 1991, supra; Wilson et al., 1992, supra; Mangelsdorf et al.,1995, supra).
There thus remains a need to identify physiological targets for the binding of Nur77 and related nuclear receptors. In addition, there remains a need to dissect the protein-protein interactions and ligand interactions relating to Nur77 and related nuclear receptors at their physiologically relevant target sites. More particularly, there remains a need to establish whether Nur family members modulate transcription as monomers and/or homodimers and/or heterodimers.
Glucocorticoids (Gc) and their receptors have been shown to repress transcription of target genes by Interaction of the Gc receptor (GR) with members of the AP-1 family of transcription factors (Jonat et al., 1990, Cell 62:1189-1204; Schüle et al., 1990, Cell 62:1217-1226; Yang-Yen et al., 1990. Cell 62:1205-1215). However, this mechanism does not account for repression of all Gc sensitive genes. This is the case of the Gc-repressed pro-opiomelanocortin (POMC) gene which encodes the precursor to ACTH, the major stimulus of adrenal Gc synthesis. Indeed, various mechanisms have been invoked to account for Gc repression of POMC transcription, including the formation of complexes containing three GR molecules on a negative Gc response element (nGRE) present at −63 bp in the POMC promoter (Drouin et al., 1993, EMBO J. 12:145-156). Some authors have suggested that a fos-dependent pathway may be involved in part but not exclusively (Boutillier et al., 1995, Mol. Endocrinol. 9:745-755) and others have implicated distal promoter sequences which are neither target for AP-1-related factors nor for GR (Riegel et al.,1991, Mol. Endocrinol.5:1973-1982).
There thus remain a need to identify the mechanism which accounts for GC repression of POMC transcription.
The present invention seeks to meet these and other needs.
The description found herein refers to a number of documents, the content of which is herein incorporated by reference.